Electrical excitation of label substances at insulating film-coated conductors

ABSTRACT

The excitation of label molecules usable in chemical and biochemical analysis by electrical pulses at electrodes covered with a thin insulating film, and the use of such electrodes in chemical, clinical and biochemical analysis. The electrodes include a conducting base material that has been coated with an organic or inorganic insulating film or multiple layers of such films, so that either one or several label compounds can be excited to an excited state which is deexcited by emission of ultraviolet, visible or infrared light, in aqueous solution providing the basis for reproducible analytical applications in bioaffinity assays such as immunoassays and DNA-probing assays.

This application is a continuation of application Ser. No. 09/341,955,filed Jul. 21, 1999 and now U.S. Pat. No. 6,251,690, which is theNational Stage of PCT/FI98/00114, filed Feb. 10, 1998 and published inthe English language on Aug. 20, 1998.

FIELD OF INVENTION

The present invention relates to electrical excitation of labelsubstances at electrodes covered with insulating layer/layers andutilisation of the resulting luminescence (electogenerated luminescence,EL) in analytical methods, especially in bioaffinity assays.

BACKGROUND OF INVENTION

Many commercially important analytical methods are bused on theprinciple that the analytes can be recognised and quantified from amatrix using label substances. For instance, in the assays based on thebiological properties of analytes, such as in immunoassays, the analyte(A) can be selectively captured from a solution upon a solid supportwith the aid of antibodies immobilised on the surface of the solidsupport, and the amount of (A) can be quantified using another antibodyselectively binding with (A) and being labelled with a suitable markersubstance. Such a marker substance can be, for instance, radioactiveisotope, enzyme, molecule that absorbs light or produces fluorescence orphosphorescence, certain metal chelates etc., which can be coupled withchemical bonds with an antibody. Alternatively, purified (A) can belabelled (A-L) and the amount of unlabelled (A) can be determined byantibodies immobilised on a solid support with by exploiting competitivereactions between (A-L) and analyte (A). DNA- and RNA-probing assays arebased on the analogous bioaffinity principles as immunoassays and can beperformed along with related procedures. Also, other chemical andbiochemical analysis methods can be based on analogous principles.Presently, there is an increasing need for multiparameter assays due toa growing demand to decease the costs and/or increase the simplicity andaccuracy of determinations. One solution to these problems is the use oflabel compounds luminescing at different wavelengths. Various methodsand strategies in immunoassays are described, e.g., in “The ImmunoassayHandbook”. Edited by David Wild. Stockton Press Ltd., New York, 1994,pages 1-618.

It is already known that organic luminophores and metal chelatessuitable for labelling in analytical methods can be excited with lightor by electrochemical means resulting in the specific emission from thelabelling substance. The methods based on these phenomena are generallysensitive and well-suited for the excitation of label substances.However, difficulties are encountered when the concentrations of labelsin real assays are very low; e.g., the use of fluorescence iscomplicated by the existence of Tyndall, Raleigh, and Raman scattering,and by the background fluorescence common in biological samples.Phosphorescence in liquid phase is mainly usable only in connection withsome specially synthesised lanthanide chelates. Utilisation of thelong-lived photoluminescence of these compounds is restricted mainly dueto complicated apparatus required and high cost of pulsed light sources.

Electrochemiluminescence can be generated in non-aqueous solvents atinert metal electrodes with a rather simple apparatus. However,bioaffinity assays which are of commercial importance are normallyapplicable in aqueous solutions only. Samples are practically alwaysaqueous and therefore the demon method of a label substance must beapplicable in aqueous solution. Presently, only certain transition metalchelates can serve as electrochemiluminescent labels in micellarsolutions, which in principle, are not fully aqueous solutions. However,these methods utilising conventional electrochemistry and inert metalelectrodes do not allow simultaneous excitation of several labelsubstances possessing sufficiently differing emission spectra and/orluminescence lifetime.

Mainly inert active metal (e.g. Pt and Au) or carbon electrodes areapplied in conventional electrochemistry. Their utilisation isrestricted to a narrow potential window due to the water decompositionreactions, hydrogen and oxygen evolution. Luminophores usable asfluoresent or phosphorescent labels cannot normally be electricallyexcited in aqueous solution at these electrodes due to theinaccessibility of the highly anodic and cathodic potentials requiredfor the excitation reactions. With suitably selected semiconductorelectrodes a wider potential window is achievable, but only very rarelabelling substances can be excited at this type of electrodes in fillyaqueous solutions.

The present invention provides considerable improvement for use ofactive metal electrodes or semiconductor electrodes and makes itpossible to simultaneously excite a variety of different labellingsubstances in fully aqueous solution. The invention utilises a new typeof electrodes, conductors covered with an insulating film, which areuseless in the field of conventional electrochemistry. Below theseelectrodes are called either insulator electrodes or insulatingfilm-coated electrodes.

SUMMARY OF THE INVENTION

This invention relates to the excitation of label molecules useable inchemical and biochemical analysis by electrical pulses at electrodescovered with a thin insulating film, and the use of such electrodes inchemical, clinical and biochemical analysis. The electrodes consist of aconducting base material that has been coated with an organic orinorganic insulating film or multiple layers of such films, so thateither one or several label compounds can be excited to an excited statewhich is deexcited by emission of ultraviolet, visible or infraredlight, in aqueous solution, thereby providing the basis for reproducibleanalytical applications in bioaffinity assays such as immunoassay andDNA-probing assays.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a measurement apparatus which incorporates theinsulating film-coated electrodes used in the method of the presentinvention.

FIG. 2 depicts the measurement principle of an immunoassay of thepresent invention.

FIG. 3 shows various shapes of the insulating film-coated electrode ofthe present invention.

FIG. 4 illustrates standard curves of an immunometric assay ofphospholipase A₂, in which the working electrodes are covered with (a) anatural oxide layer, (b) an anodized oxide layer, and (c) an anodizedoxide layer covered with polystyrene.

FIG. 5 depicts standard curves of an immunometric assay of TSH in whichthe working electrodes are aluminum electrodes covered with (a) naturaloxide layer, (b) a layer modified by anodization and by coating with anepoxy plastic layer.

FIG. 6 shows a standard curve of a competitive assay of thyroxine (T4)in which the insulating film-coated electrodes were anodized aluminumelectrodes.

FIG. 7 illustrates the detection of Philadelphia chromosome by a DNAhybridization method in which the insulating film-coated electrodes werealuminum electrodes coated with polystyrene.

FIG. 8 depicts standard curves of an immunometric assay of TSH in whichthe electrodes were of oxide-coated aluminum (a), and (b) the surface ofthe oxide was modified by silanization.

FIG. 9 shows a standard curve of an immunometric assay of CRP in whichthe insulating film-coated electrodes were oxide-covered magnesiumelectrodes coated with polystyrene.

FIG. 10 illustrates a standard curve of an immunometric assay ofβ₂-microglobulin whereby the insulating film-coated electrodes wereanodized silicon electrodes.

FIG. 11 depicts standard curves of an immunometric assay of TSH in whichthe working electrodes were (a) silicon electrodes covered with anatural oxide film, (by anodised silicon electrodes, and (c) anodizedsilicon electrodes covered with polystyrene.

FIG. 12 shows the detection of Philadelphia chromosome by a DNAhybridization method in which the insulating film-coated electrodes wereanodized silicon electrodes covered with polystyrene.

FIG. 13 illustrates a standard curve of a competitive assay of thyroxine(T4) in which the insulating film-coated electrodes were anodizedsilicon electrodes.

FIG. 14 depicts standard curves of an immunometric assay of TSH wherebythe insulating film-coated electrodes were zinc electrodes either (a)first treated cathodically, or (b) directly coated with sequentiallayers of polystyrene and paraffin.

FIG. 15 shows a standard curve of an imrnunometric assay ofβ₂-microglobulin when the insulating film-coated electrodes wereITO-glass plates coated sequentially with polystyrene and paraffin.

FIG. 16 illustrates a standard curve of an immunometric assay ofβ₂-microglobulin whereby the insulating film-coated electrodes wereAu-PET films coated sequentially with polystyrene and paraffin.

FIG. 17 depicts a standard curve of an immunometric assay of CRP wherebythe insulating film-coated electrodes were polyaniline films coatedsequentially with polystyrene and paraffin.

FIG. 18 shows a standard curve of an immunometric assay of CRP wherebythe insulating film-coated electrodes were of steel coated withaluminium oxide and polystyrene.

FIG. 19 illustrates a recorded EL spectra showing simultaneousexcitability of a short-lived and long-lived EL-emitting labels.

FIG. 20 depicts standard curves of simultaneous immunometric assays ofTSH and PLA₂ whereby the insulating film-coated electrodes were anodizedaluminum electrodes coated with polystyrene.

FIG. 21 shows a standard curve of an immunometric assay of phospholipaseA₂ using latex beads whereby the insulating film-coated electrodes wereanodized aluminum electrodes coated with polystyrene.

FIG. 22 illustrates standard curves of immunometric assays ofphospholipase A₂ whereby enzymatic amplification was applied measuringdirectly 5-fluorosalicylic acid-label (a), and by producing ternaryTb(III) complex prior to EL measurements, applying total EL(b) ortime-resolved EL detection (c) with polystyrene-coated magnesiumelectrodes.

FIG. 23 depicts a standard curve of an immunometric assay ofβ₂-microglobulin whereby the insulating film-coated electrodes were madeof anodized silicon.

FIG. 24 shows a standard curve of an immunometric assay ofβ₂-microglobulin whereby the insulating film-coated electrodes were madeof anodized silicon and the label was composed of liposomes containingluminophores.

FIG. 25 illustrates a standard curve of an immunometric assay ofβ₂-microglobulin whereby the insulating film-coated electrodes were madeof anodized silicon and the label was a luminophore that can bephotodetached by UV-light.

FIG. 26 depicts the standard curve of an immunometric assay ofβ₂-microglobulin based on energy transfer whereby the insulatingfilm-coated electrodes were made of anodized silicon.

FIG. 27 shows a standard curve of an immunometric assay ofβ₂-microglobulin whereby the insulating film-coated electrodes were madeof anodized silicon and the label was composed of polystyrene particlescontaining terbium chelates.

DETAILED DESCRIPTION OF THE INVENTION

The aim of the invention is a method and apparatus, with which one orseveral different types of label substances can be simultaneouslyelectrically excited, so that the resulting luminescence can be utilisedin bioaffinity assays such as immunoassays and DNA or RNA probingassays.

It has been experimentally observed that extremely harsh redoxconditions can be produced at aluminium electrodes, and that theseconditions closely resemble those of radiolysis of water (S. Kulmala,“Electrogenerated lanthnide(III) luminescence at oxide-covered aluminiumelectrodes and closely related studies”, Academic dissertation, Turunyliopisto, 1995). Electrically induced luminescence at aluminiumelectrodes has already been studied for several years usingirreproducible results-yielding electrodes with natural oxide filmcoverage as described e.g. in references: J. Kankare, K. Fälden, S.Kulmala and K. Huaapakka, Anal. Chim Acta, 256 (1992) 17. and J.Kankare, K. Haapakaa, S. Kulmala, V. Näntö, Eskola and H. Takalo, Anal.Chim. Acta, 266 (1992) 205. The nature of the metal itself was assumedto be the most important component of the system and the importance ofthe naturally existing 1-2 nm thick oxide film was not understood. Forinstance, in the life, tantalum Electrodes were claimed to be fullyequivalent with album electrodes and usable in the same applications asaluminium electrodes (UK Patent GB 2 217 007 B). However, tantalum oxideis an n-type semiconductor with a band gap ca. 4 eV (S. Morrison,“Electrochemistry at Semiconductor and Oxidised Metal Electrodes”,Plenum Press, New York, 1980, s.183) and, therefore, oxide coveredtantalum electrodes cannot be used according to the principles of thepresent invention. In the present invention, insulator electrode isdefined as an electrode on which at least one of the coating layersconsists of material that has band gap larger than or equal to 5 eV.

The present invention is based on a thin, normally close to 4 nm thick,good-quality insulating film, upon the surface of which or in thevicinity of which the bioaffinity reactions are performed, or to thevicinity of which the products of the bioaffinity reaction are broughtwith a suitable medium such as electrolyte solution, or upon suitablesupporting material such as surface of magnetic latex particles. Theapplicability of the present invention is partially based on the factthat the existence of an insulating film enables the Fermi-level of thebase conductor to reach highly cathodic pulse potentials, andsubsequently allows a transfer of energetic (hot) electrons into theelectrolyte solution, either by tunnelling through the insulating filmor as a consequence of an electron avalanche, If the Fermi level of theelectron-emitting base conductor is above the conduction band edge ofwater (−1.3 eV on vacuum scale), the hot electrons can be injected intothe conduction band of water and thus produce hydrated electrons ascathodic mediators for reduction reactions as has been described in thecases of radiolysis of water or photoionisation of solutes.

The insulating film on the electrodes also provides the basis for theFermi level of the base conductor to reach highly anodic pulsepotentials, which makes a new anodic process, a hole injection into thevalence band of water, possible. This process is analogous to theelectron injection into the conduction band of water, and results in thegeneration of hydroxyl radicals by dissociation of H₂O⁺-ion formed(valence band hole in the water) to proton and hydroxyl radical as knownfrom the pulse radiolysis of water. Certain metal oxides, Al₂O₃, SiO₂,and MgO, may produce hydroxyl radical also by other solid statemechanisms as described in references: S. Kulmala, T. Ala-Kleme, A.Kulmala, D. Papkovsky and K. Loikas, “Cathodic ElectrogeneratedChemiluminescence of Luminol at Disposable Oxide-covered AluminumElectrodes”, Anal. Chem., in press.; S. Kumala & T. Ala-Kleme, Anal.Chim. Acta, 355 (1997) 1-5.

Hydroxyl radical, having a strong tendency to addition and hydrogenabstraction reactions, can be transformed into other oxidising radicalswhich are better suited reactants in producing redox luminescence. Thesesecondary oxidising radicals can be produced by addition of anions fromthe halide, and pseudo-halide series into the measuring electrolytesolution (X⁻=halide or pseudo-halide-ion);

OH+X⁻→OH⁻+X⁻

If an insulating film is incapable of producing hydroxyl radicals by theabove-mentioned mechanisms or if one wishes to increase the amount ofoxidising species at the expense of reducing equivalents, hydroxylradicals can be generated from hydrogen peroxide according to reaction:

H₂O₂+e⁻→OH+OH⁻

An analogous technique also allows the production of sulfate andphosphate radicals, which are often better suited oxidants for the redoxexcitation pathways than hydroxyl radicals (S. Kulmala T. Ala-Kleme, A.Kulmala, D. Papkovsky and K. Loikas, “Cathodic ElectogeneratedChemiluminescence of Luminol at Disposable Oxide-covered AluminumElectrodes”, Anal. Chem, in press.):

S₂O₈ ²⁻+e⁻→SO₄ ⁻+SO₄ ²⁻

P₂O₈ ⁴⁻+e⁻→PO₄ ²⁻+PO₄ ³⁻

The protonation of phosphate radicals affect the oxidising power of theradical whereas the oxidising power of sulfate radical is independent ofthe pH above pH 2.

Hence, highly oxidising and reducing conditions can be createdsimultaneously in the vicinity of insulating film-coated electrodes,which is usually a prerequisite for the existence of redox luminescencein aqueous solution.

Although the solid state phenomena utilised in this invention are knownin the theories of physics, the present type of insulator electrodes hasnot been utilised in analytical chemistry, except in the case of albumelectrodes covered with naturally existing poor-quality and too thinoxide film (UK Patent GB 2 217 007 B) that have not led to any practicalapplications of such aluminium electrodes. On the contrary, the presentinvention forms a major improvement of the said electrodes by realisingthe correct role of the insulating films and their deliberatepreparation.

According to the present invention, electrodes have a conductive baselayer that can be composed of e.g., carbon (graphite, glassy carbon) ormetal such as Be, Mg, Al, Ga, In, Au Pt, Cu, Fe, Ru, stainless steel,Zn, Hg, Ag, Ni, Pd, Hf, Zr, (also Ta is suitable as the base conductoralthough Ta₂O₅ is not usable as an insulating film). However, it ispossible that the electrode works better the smaller the work functionof the base conductor is.

The conductor can be also a heavily doped semiconductor or metal oxidesuch as Si, Ge, Sr, ZnO, SnO₂ etc. The base conductor can be alsocomposed of conductive polymer such as polyaniline, polypyrrole,polyacetylene, polytiophene or of corresponding polymers made fromsubstituted monomers. The resistivity of the base conductor should be<10 Ωcm.

Insulating layer(s) of the electrode can be made of some metal oxides,such as, SiO₂, MgO, CaO, SrO, BaO, Al₂O₃, HfO₂; of some other inorganicinsulators, such as diamond, silicates or nitrides, some organicinsulating materials such as paraffines, other solid or liquidhydrocarbons, organic insulating polymers such as, Teflon, polyethene,polypropene, polystyrene, polyacrylamides, epoxy-plastics etc. Normallymetal oxides can be used as insulating film material only in the case ofutilisation of pulsed excitation, because DC-cathodisation normallyruins the insulating properties of the oxide films within fewmilliseconds.

Coating film of the electrodes can be manufactured by anodic oxidation,by Atomic Layer Epitaxy (ALE), by spraying polymeric or polymerisablematerial on the surface of the electrode, by dipping the electrodes inthe above-mentioned solution and letting the solvent evaporate, byLangmuir-Blodgett methods or by other methods known from other coatingprocesses. Especially in the case of silicon, there are severalalternative methods to manufacture good-quality SiO₂ films known in theelectronics industry.

Although, in principle, naturally existing oxide film-covered aluminumelectrodes can be used to excite some label substances, the commercialutilisation of these electrodes is impossible due to the poor quality ofthe natural oxide film which results in the too high irreproducibilityof the analysis results (S. Kulmala, “Electrogenerated lanthanide(III)luminescence at oxide-covered aluminium electrodes and closely relatedstudies”, Academic dissertation, Turun yliopisto, 1995, pp. 25-31 and114-119.) However, the reproducibility of the analysis can be improvedto the level required, by fabrication of good-quality insulating filmwith suitable thickness. It is characteristic for the present inventionthat the coating film/films are carefully layered upon the baseconductor taking care that the total thickness of coating is opt . Inthe case of aluminium, this kind of insulating film cannot be producedby letting aluminium to be oxidised in air, but can be manufactured byother methods. A preferred method is anodic oxidation of aluminium insuitable electrolyte solution and successive coating with organicmaterial(s) to prevent the aqueous solution spoiling the insulatingproperties of the oxide film during the bioaffinity assays that isalways inevitable to some degree in the absence of other shieldingcoating layers. Aluminium oxide film can also be made by coating someother material with aluminium, such as plastic, graphite, glassy carbon,metal, and subsequently oxidising aluminium and adding a final shieldinglayer.

When an aluminium oxide film is immersed in an aqueous solution,depending on the solution conditions, various uncontrollable processesstart to proceed in the oxide film commencing from the outer part of thelayer. Consequently, the properties of the naturally existing very thin(1-2 nm thick) spontaneously formed oxide film change as a function oftime and induce strong decrease in the EL generation efficiency, Aspointed out in Example 1, in aqueous solution the EL generationefficiency decreases 90% dung coating with a protein film necessary asthe first step of any immunoassay. This drawback can be prevented, whenthe oxide film has been fabricated to optimal thickness and preferablycovered with an additional shielding thin film, such as an organicinsulating polymer film. Some organic polymers are also especiallybeneficial because they improve the coatability of the electrodes withantibodies and other biomaterials.

In bioanalytical methods, DNA- and RNA-probing techniques arepractically as important techniques as immunoassays. Aluminiumelectrodes covered with thin naturally existing oxide film arcunexpectedly not at all applicable in nucleic acid hybridisation assays.However, it was experimentally found out that even aluminium. eletrodesbecome useful also in these methods, if the oxide film is coated with athin organic film such as polystyrene layer.

Suitable polymeric films can be readily created by dipping the electrodein polystyrene solution which has been made by dissolving polystyrene inan organic solvent such as benzene or toluene. In an analogous way, alsomany other polymers that can be dissolved as dilute solutions ofsolvents can be utilised in fabrication of thin polymer films. Amongusable polymers there can be mentioned: polyamides, polyamines,polybutadienes, polycarbonates, polyenes, polyesters, polyethylenes,polyethyleneimides, polyformaldehyde etc. as has been described in thetextbooks of polymer chemistry. Polymeric films can also be made frommixtures of polymers or they can be doped with inorganic materials.Polymeric film can be made exceptionally smooth by using commerciallyavailable apparatuses for growing of these films. The electrode surfaceneed not to be totally coated with an insulating film designed foractive use in excitation of labels, but insulating films can be in theform of very small spots or islands, surrounded by a thicker insulatingfilm not allowing current transport with any mechanism. The electrodesare not necessarily always plate-like, but can also have a shape of anet, spike(s), tube(s), a plate with hole(s), etc.

Analogous polymeric films can be created also by allowing polymerisationto occur at the surface of a conductor or at the surface of insulatingfilm-covered conductor. In this case, the reactants of polymerisationreaction ate dissolved separately in a suitable inert solvent such astoluene, benzene, dichlormethane, etc. One of the components can bedeposited with a special deposition device, or the electrode isautomatically dipped in a solution of the component and solvent isallowed to evaporate. Another component can be deposited in an analogousway and polymerisation is allowed to occur. Alternatively, the reactantsof polymerisation reaction are mixed in a solvent just beforedeposition. In all cases, the optimal thickness of the films can beexperimentally found by adjusting the concentration of coating materialsin the solvents or by using coating apparatus for adjustment of the filmthickness. In some cases it is preferable to coat electrodes byspraying. Polymer must be divided in the spray to droplets with diameter1-1000 nm in an appropriate solvent, such as, toluene, benzene,cyclohexane, chlorinated hydrocarbons, DMF, DMSO, or alcohols.

The main drawback of aluminium oxide films is that they cannot tolerateeither basic or acidic conditions. This drawback can be prevented by anextra shielding layer or replacing aluminium oxide films with otherinsulating films. MgO films are suitable especially in basic conditionsbecause these films are not dissolved in basic aqueous solutions. MgOfilms can be made e.g. by ALE-techniques and also these films can becoated with other films as pointed out above in the case of aluminiumoxide films.

Al₂O₃, MgO and other alkaline earth metal oxide coatings are lessstudied than rather well known SiO₂ films. SiO₂ is the most importantinsulating film material in electronics industry based on silicontechnologies. There are several methods available for fabrication ofgood-quality SiO₂ films in this field of industry. Therefore,technically mature silicon technology is preferable in the fabricationof the electrode materials utilised in the embodiment of this invention.

Inorganic insulating films can be totally replaced with suitable organicinsulating films, if the production of oxidising species, generallynecessary for excitation most of the label substances, is provided byaddition of suitable coreactants such as peroxydisulfate,peroxydiphosphate, or hydrogen peroxide which produce strongly oxidisingradicals via one-electron reduction. Often, the combination of inorganicand organic film is preferable.

The present invention makes a dramatic improvement over the prior artspecially, with increasing the reproducibility to a level required inpractical analytical methods. Other advantages of the present inventionare the excitation event itself and the accurate timing of excitation.In addition, a number of very differing label substances (emissionspectra and luminescence lifetimes of which are different) can besimultaneously excited which allows multiparametric assays Example 17).They also render a possibility to improve accuracy of determinations byinternal standardisation. For instance, in homogeneous assays whereunreacted excess label is not separated from immunocomplexes, duallabelling enables to quantitate simultaneously two different antibodiesor their concentration ratio (Example 23). This allows efficientexclusion of matrix effects arising from deviating sample compositionsoften preventing the exploitation of homogeneous assays. Other majoradvantages of the present invention is the simplicity and low cost ofthe measuring instrument.

According to the present invention many types of luminophores can serveas the labels. For instance, the following luminophores (or derivativesof them) can be utilised: 9-fuorenylmethylchloroformate (emission 309nm), luminol (emission 420 mn), fluorescein (emission 516 nm),salicylates (emission in the region 400-450 nm), aminonaphthalenesulphonates (emission in the range of 400-500 nm) and coumarines(emission in the range of 450 nm-550 nm), aromatic lanthanide(III)chelates, such as certain derivatives of terbium(III) complexes withfollowing ligands:N¹-(4-aminobenzyl)diethylenetriamine-N¹,N²,N³,N³-tetra-acetate(Tb(IIl)-1),4-(phenyl-ethyl)(1-hydroxybenzene)-2,6-diyl)bis-methylenenitrilo)tetrakis(acetate)(Tb(III)-2);4-benzoyl(1-hydroxybenzene)-2,6-diyl)-bis(methylenenitrilo)tetrakis(acetate)(Tb(IlI)-3),N²-(4-aminobenzyl)-diethylenetriamine-N¹,N¹,N³,N³,-tetra-acetate(Tb(III)4);4-methyl(1-hydroxybenzene)-2,6-diyyli)bis(methylenentrilo)tetakis(acetate)(Tb(III)-5)(The strongest emission line at 545 nm in the cases of allTb(III) chelates), derivatives of certain transition metal chelates suchas ruthenium(II) and osmium(II)-trisbipyridyl and trispyratsyl complexes(emission in the range of 550-650 nm).

A high electric field across the insulating films induces also to someextent solid state electroluminescence in the film. This solid stateelectroluminescence makes possible the excitation of luminophores alsoby energy transfer from the intrinsic emission centres, if theluminophores are located sufficiently close to the insulating film. Thiseffect enhances the proximity effect required by homogeneous assays.

The insulating film-coated electrodes described in the present inventioncan be used in an EL cell which contains at least two electrodes: aninsulating film-covered working electrode and a counter electrode.

The insulating film-coated working electrode should fulfill the criteriadescribed above, and depending on the optical properties and thethickness of the conducting material it can be either opticallytransparent or non-transparent. Usually the transmittance of thesufficiently thin base conductor is high enough in the desired opticalrange. The use of a transparent working electrode makes possible themeasurement of electrically excited luminescence through the workingelectrode.

The selection of the counter electrode of the method is not critical.Conventional inert electrode materials (Pt, Au) are well suited. Often,even certain metals which are anodically dissolved can be used, becausethe measurements usually are made in the time scale where the anodicproducts from counter or auxiliary electrode do not have time to diffuseto the working electrode. Also some metal oxide electrodes, such asindium tin oxide, are well suited as an anode material. In this case theanode material can be readily made optically transparent. Stainlesssteel is also advantageous electrode material. If a non-transparentmetal electrode serves as a counter electrode its shape can be chosen sothat the luminescence is measurable behind the counter electrode. Forinstance, a wire electrode covering only a very small part of thesurface of the working electrode can be used or hole(s) drilled throughthe anode material allow the light detection behind the anode. Opticallytransparent counter electrode can be prepared from ad equate film, e.g.,from plastic or glass coated with a thin Au-film which can be furthercoated with a thin shielding film allowing electron and/or holetunnelling through the outer film.

If the thickness of the insulating film on the working electrode issuitable, the excitation of the label in the detection stage can be doneby cathodic voltage pulse train, but in the case of base material beinganodically oxidisable material such as Si, Al, Be or Mg, it is sometimesbeneficial to grow the oxide film thicker by oxidising anodic pulsebefore each cathodic excitation pulse.

Depending on the sensitivity range needed for the analyte(s), either alow-cost semiconductor detector or more expensive and more sensitivephotomultiplier tube are suitable light detectors for theelectroluminometer.

The method of the present invention may be used to detect a moleculehaving the form L_(n), -X_(x)-Y_(y), where L is label or mixture ofdifferent kinds of labels, the label being a derivative of an organicluminophore, like a derivative of fluorescein, aminonaphthalenesulphonicacid, salicylates, rhodamines, or coumarines; a derivative of lanthanidechelates, like a derivative of Tb(III), Eu(III), Y(III), Sm(III),Dy(III), Gd(III) chelates; a derivative of transition metal chelates,like ruthenium(II)-trisbipyridyl- or ruthenium(II)-trispyrazyl chelates;where one or more of the derivatives are bound through suitablefunctional groups either directly or through one or more of linkingcompounds X to the compound Y, where Y is exemplified as protein,antibody, enzyme, or nucleic acid, which have affinity against theanalyte to be quantified, and wherein the integral subscripts n, x, andy express the number of L, X, and Y, and are equal or larger than 1, andwhere compound Y can bind to a cell, a cell component, a virus,bacterium, nucleic acid, DNA, RNA, DNA-fragment, RNA-fragment,polysaccharide, protein, polypeptide, enzyme, metabolite, hormone,pharmacological substance, medical drug, alkaloid, steroid, vitamin,amino acid, carbohydrate, environmental pollutant, or antibody, andwhere the joint compound X may contain, as the essential linkingfunction, a chemical group such as ureido, thioureido, amide,substituted imide, thioether, —S—S—, sulfonamide, or N-substitutedsulfonamide, that is a part of a larger molecule or polymer attaching tothe compound Y.

The method of the present invention includes a competitive bioaffinitymethod, where a competition of binding to Y on the surface of aninsulating film-coated electrode is created between labelled analyteL_(n)-X_(x)-A_(a) where the integral subscripts n, x and a express thenumber of L, X and A, and analyte A originated from the sample enablingthe analyte concentration to be determined with insulating film-coatedelectrodes. Binding of L_(n)-X_(x)-Y_(y) to A on the surface of theinsulating film-coated electrode may inhibited by A originated from thesample.

The label L may be an enzyme capable of amplification of luminescentluminophores. For example, the enzyme may be alkaline phosphatase andthe luminescent molecule may be a highly luminescent molecule or alanthanide chelate generated by the enzyme.

The method of the present invention includes assays (i) in which onlythe label molecules located in the proximity of the insulatingfilm-coated layer film can be excited by the electrical pulses enablingthe analysis to be carried out by the homogeneous assay principle, andthe separation of free label L_(n)-X_(x)-Y_(y) is not required beforethe detection step, (ii) the quantitation is performed withheterogeneous principle and the free label L_(n)-X_(x)-Y_(y) is removedfrom the proximity of electrode by a washing step before the detectionstep, and (iii) the basic immunoreaction is carried out in a separateincubation chamber with small-sized solid support materials, such asparamagnetic latex particles, whereby only the detection of the label iscarried out on the electrode surface, after incubation and possiblewashings, by bringing the solid support materials into the proximity ofthe electrode.

The novel electrodes described in the present invention can be used toelectric excitation of also other kind of luminophores than presented inDescription and Examples of the present invention because it is obviousthat also many other kind of molecules can be excited at insulatingfilm-coated electrodes with the present methods. The use of insulatingfilm-covered electrodes is not limited to certain equipment constructionor just to analytical methods described hereby. In principle, also thereverse processes can be utilised as well. In this case, label moleculesare illuminated with light and the resulting photocurrent induced by thephotoinjection of carriers into probing electrode is measured or thepotential of the probing electrode is measured allowing the quantitationof labels and analytes. In these applications, the probing electrodeshould preferably not be covered with an insulating film or favourablythe thickness of the passive film should be less than 4 nm.

EXAMPLE 1

Preparation of insulator electrodes by anodising aluminium and modifyingthe surface by coating with polystyrene, and the immunoassay ofphospholpase A₂ on the surface of these modified electrodes.

Anodic oxidation of Al-electrode. Al-electrode (Merck Art. 1057) mouldedand cut to the final shape was first washed in ultrasound bath withhexane. Hexane was then allowed to evaporate. The electrodes were firstoxidised galvanostatically with current density of 2 mA/cm² in 0.5 mol/Lboric acid solution neutalised with ammonia until the anodising voltageof 2.92 V was reached. After this the anodising was continuedpotentiostatically until the current density was less than 10 μA/cm².

Coating oxidised electrode with polystyrene. The anodised parts ofelectrodes were coated first with polystyrene by sonicating theelectrode for 10 s in the solution containing 0.7 mg/mL of polystyrenein benzene. Next the electrode was slowly lifted from the solution andallowed to dry at room temperature. The dry electrode was then slowlyimmersed into above-mentioned polystyrene solution further twiceallowing the solvent totally to evaporate at room temperature betweeneach immersion.

Coating electrode with antibody. Typically electrodes were coated with amonoclonal antibody by incubating the electrode overnight in TSA-buffer(0.05 mol/L Tris-HCl, pH 7.75, 0.9% NaCl, 0.05% NaN₃) containing theantibody (10 μg/mL; anti-PLA₂, clone 2E1, Labmaster Oy, Turku, Finland).Next day the electrode was washed six times with wash solution (0.01mol/L Tris-HCl, pH 7.75, 0.9% NaCl and 0.02% Tween 20) and equilibratedovernight in saturation solution (TSA-buffer containing per liter 1 gbovine serum albumin 60 g sorbitol and 1 mmol CaCl₂). The equilibratedelectrode can be dried at room temperature without washing and storeddry at least for 3 months.

Preparation of labelled antibody. The polyclonal sheep antibody specificto human pancreatic phospholipase A₂ (affinity purified by Labmaster Oy,Turku, Finland) was labelled with an isothiocyanate derivative ofTb(III)-1 chelate [Tb³⁺-N′-(p-isotiosyanatobenzyl)diethylene triame-N¹,N², N³, tetra acetate], (Wallac Oy, Turku, Finland) by allowing theantibody to react with the chelate in the molar ratio of 1:60 at pH 9.5.The pH was adjusted with 1 mol/L Na₂CO₃ solution. The labelled antibodywas separated from unreacted chelate by gel filtration (Sepharose 6B1×50 cm, Sephadex G-50 1×5 cm) using TSA-buffer as eluent. Typically5-10 chelate molecules can be bound in this way to one antibodymolecule. To improve the stability, 0.1% bovine serum albumin was addedto labelled antibody.

Preparation of standards. The standards of human PLA₂ (0, 1, 5, 9, 54,324 ng/mL, Labmaster Oy, Turku, Finland) were prepared in TSA-buffercontaining 7% bovine serum albumin.

Immunoassay. The immunoassay was carried out in the wells of microtiterstrips. First, 25 μL of standard and 175 μL assay buffer (0.05 mol/LTris-HCl, pH 7.75, 0.9% NaCl, 0.5% NaN₃, 0.5% bovine serum albumin,0.05% bovine gammaglobulin and 0.01% Tween 40) were added. Next theelectrode was added and after incubation for 1 h the electrode waswashed with g wash solution and allowed to react with labelled antibody(500 ng/200 μL) for 1 h. After the reaction, the electrode was washedand the EL was measured in the measuring solution (0.2 mol/L boratebuffer, pH adjusted to 7.75 with sulphuric acid) containingalternatively either 0.01 mol/L NaN₃ or 1 mmol/L K₂S₂O₈. FIG. 4 showstypical standard curves obtained in the assays, where (a) electrodeswere air-oxidised, (b) electrodes were anodised with 2.92 V, and (c)electrodes anodised with 2.92 V and coated with thin polystyrene layeras described above (in the meaning solution of 1 mol/L K₂S₂O₈). Themeasurements were performed using aluminium cup electrodes and theinstrument described in an academic dissertation of S. Kulmala(University of Turku, Tuku, Finland, 1995, pp. 34-35). In themeasurement the excitation pulse was 200 ms and −10 V with the frequencyof 100 Hz. The intensity of EL was integrated during 200 excitationpulses.

EXAMPLE 2

Modifying surface of an insulator electrode by coating with epoxy resinand immunoassay of TSH on the surface of these modified electrodes.

The electrodes were cleaned and anodised as described in Example 1 andthe electrodes were covered with thin layer of epoxy resin.

Coating electrodes with epoxy resin. Both components of the Super epoxyglue (Loctite Finland Oy, art. n:o 120-1, Finland) were dissolved 1%(w/v) in toluene. The components were mixed 1:1 and the electrode wasimmersed into this solution in ultrasound bath and then lifted slowlyout of this solution. The flowing solution from the lower edge was driedand the electrode was allowed to dry at room temperature until toluenewas evaporated and then further in an oven at 40° C. for 24 h. Thickercoatings can be made by repeating the procedure.

Coating electrode with antibody. Electrodes were coated with antibody byphysical adsorption by incubating the electrode in TSA-buffer containingthe coating antibody (clone 8661, specific to the alpha chain of TSH,Pharmacia, Uppsala, Sweden) 30 μg/mL for 3.0 h. After the coating thesurface was washed with running wash solution and equilibrated overnightin TSA-buffer, pH 7.75, containing 0.1% bovine serum albumin and 5%D-sorbitol. After the equilibration the electrodes were dried and theywere stable in storage for at least one year.

Preparation of labelled antibody. The labelled antibody for the assay ofTSH was prepared in the same way as in Example 1. In this case themonoclonal antibody that is specific to the β-chain of TSH (clone 5404,Medix Oy, Helsinki, Finland) was labelled.

Preparation of standards. TSH standards (0, 0.25, 1.5, 9, 54, 324 μU/mL)were prepared by diluting the stock standard (Scripps Laboratories Inc,San Diego, USA) in TSA-buffer containing 7% bovine serum albumin.

Immunoassay. TSH standard (20 μL) and 180 μl (300 ng) of labelledantibody were added to the polystyrene wells. After incubation for 1 h,the electrode was washed and the EL was measured as in Example 1a-1b,but using the electroluminometer constructed by modifying an Arcusfluorometer (Wallac, Turku, Finland) The rake electrodes were as in FIG.3c. FIG. 5 displays steward curves obtained with electrodes covered (a)with naturally exiting oxide film, and (b) with electrodes covered withan anodic oxide film and an epoxy film.

EXAMPLE 3

Competitive immunoassay of thyroxine (T4) whereby the insulatorelectrodes were anodised aluminium electrodes covered with polystyrene.

Labelling of thyroxine. Thyroxine was bound to gelatine as follows.T4-N-hydroxysuccinimide ester (1 mg, Wallac Oy, Turku, Finland) wasdissolved in 194 mL of dioxane and 0.1 mL of this solution was added to1 mL of 0.05 mol/L phosphate buffer, pH 7.3, containing 20 mg of gelatin(E. Merck, Darmstadt, Germany). After the reaction overnight at +4° C.gelatin was separated from small-molecular reagents by gel filtrationusing PD10-column (Pharmacia, Uppsala, Sweden) and phosphate buffer aseluent as above. The purified conjugate was labelled with isothiocyanatederivative of terbium chelate as in Example 1, but in this case themolar ratio of Tb-chelate to gelatin was 200:1. By assumption that themolecular weight of gelatin is 1 million it is possible to bind about100 Tb-chelates to one gelatin molecule.

Coating electrode with antibody. Electrodes were coated with rabbitanti-mouse immunoglobulin (Dako, Glostrup, Denmark) by incubating theelectrode overnight in 0.1 mol/L phosphate buffer, pH 4.9, containingthe antibody 5 μg/mL. Next day the electrode was washed with washsolution as in Example 1 and equilibrated overnight in TSA-buffercontaining 0.1% bovine, serum albumin, 6% D-sorbitol and 1 mmol/L CaCl₂The coated electrode was dried without washing. It could be stored atroom temperature without loss of activity for at least 3 months.

Preparation of standards. The standard of T4 (0, 10, 50, 100, 150 and300 nmol/L) were prepared in 0.01 mol/L HEPES-, 0.001 mol/L sodiumphosphate buffer, pH 7.4, containing 0.1 mol/L NaCl and 0.1% NaN₃(HEPES-buffer), and 0.1% casein.

Immunoassay. To the wells of microtiter plates 20 μL of standard werepipetted with 100 μL (0.5 ng) monoclonal mouse anti-T4 antibody (MedixInc, USA) and 100 μL of gelatin-T4-Tb-conjugate (100 ng/mL). Theelectrode was added to the well and after incubation for 1 h theelectrode was washed 5 times and the measurement was done as in Example2. The standard curve is shown in FIG. 6.

EXAMPLE 4

Detection of Philadelphia chromosome by DANA hybridisation methodwhereby the insulator electrodes were made of anodised aluminium andcoated with polystyrene.

Labelling the probe with Tb-chelate. The oligonucleotide containingamino groups (TTCGGGAAGTCGCCGGTCATCGTAGA-(C-NH₂)₂₅-5′, Wallac, TurkuFinland) was labelled with Tb-chelate as in Example 2 except that thelabelled nuclectide was purified using NAP-5 and NAP-10 columns(Pharmacia, Uppsala, Sweden).

Labelling the probe with biotin. For the assay, also another probe(C-(NH2-C)-GTCGTAAGGCGACTGGTAGTTATTCCTT-5′, Wallac, Turku, Finland) wasprepared. The N-hydroxysuccimide derivative of biotin was allowed toreact in 3.7 μL of N,N-dimethyl formamide overnight with the probe (5nmol in 50 μL, in molar ratio of 50:1) at pH 9.5, at +4° C. pH wasadjusted by adding Na₂CO₃ so much that the fine molarity was 50 mmol/L.The probe was purified as the one labelled with Tb-chelate.

Coating electrode with streptavidin. The electrode was coated byincubating for 12-15 h in TSA-buffer containing streptavidin 10 μg/mL.Next day the electrode was washed with wash solution and equilibratedovernight in TSA-buffer containing 0.1% NaN₃, 0.5% bovine serum albuminand 6% sorbitol overnight. The electrode was dried and stored dry.

Hybridisation. A sequence of 170 base pairs, from K562 human cell line,amplified with PCR from chromosome Ph¹ (TYKS, Turku, Finland) was usedas a positive sample and distilled water was a negative control. Thepositive sample and the negative control were kept in 100° C. for 10minutes and then cooled down on an ice bath after which they werecentrifuged with a microcentrifuge for 1 minute at 12000 rpm. Sample (50μl) was pipetted into a disposable cuvette followed by 200 μl of theassay buffer, which contained 2 ng of biotinylated probe and 2 ng of theprobe labelled with Tb(III) chelate. One liter of assay buffer (25 mMTRIS-HCl, pH 7.75) conned 33.72 g of NaCl, 0.25 g of NaN₃, 2.5 g of thebovine serum albumin, 0.25 g bovine serum gammaglobulin, and 0.05 mLTween 40. The reaction was allowed to proceed for 2 hours at 50° C.After the electrode was washed 6 times, EL was measured as in Example 2.FIG. 7 shows the results achieved with electrodes uncoated (a) andcoated with polystyrene (b).

EXAMPLE 5

Modifying the surface of aluminium electrodes by silanisation and theimmunometric assay of TSH on the surface of these modified electrodes.

Silanisation of the aluminium electrode. The oxide-covered aluminiumelectrode was first washed with toluene in ultrasonic bath and thendried at 100° C. for 1 hour Silanisation was carried out by sonicatingthe electrode twice for 30 seconds in a 5-% toluene solution ofdicloromethylsilane. After the silanisation the electrode was rinsedonce with toluene and twice with methanol.

Coating electrode with antibody. The electrode was coated as in Example2.

Preparation of labelled antibody Anti-TSH antibody was labelled with theisothiocyanate derivative of Tb(III)-2-chelate as shown in Examples 1and 2.

Preparation of standards. TSH standards were prepared as in Example 2.

Immunochemical determination. The immuochemical determination andmeasuring of the EL were cared out as in Example 2. The standard curvesachieved in the tests are shown in FIG. 8.

EXAMPLE 6

Immunochemical determination of C-reactive protein by magnesiumelectrodes modified by coating the magnesium oxide surface of theelectrode with polystyrene.

Coating electrode with polystyrene. A suitably sized Mg electrode (MerckArt. 5812) was washed in hexane using ultrasonic bath. Hexane wasallowed to evaporate and the electrode was coated with polystyrene as inExample 1.

Coating electrode with antibody. The electrode was antibody-coated withmouse anti-human CRP antibody (clone 7H4, Labmaster Ltd., Turku) byincubating it for 1 hour in TSA buffer (pH 8.7) containing 10 μg/mL ofantibody. The electrode was washed, stabilised and stored as in Example1.

Preparation of labelled antibody. The antibody (House anti-humanC-reactive protein, CRP, clone 5F3, Labmaster Ltd., Turku) was labelledas in Example 1.

Preparation of standards. Standards (0, 5, 50, 500, 2000 and 5000 ng/mL)were made by dissolving stock solution of CRP (Labmaster Ltd., Turku) inTSA buffer containing 7% bovine serum albumin and 1 mmol/L of CaCl₂ (thestandard buffer).

Immunochemical detection. 10 μL of standard and 200 μL of standardbuffer were added into a disposable cuvette (Brad, Cat Number 7590 15,Wertheim, Germany) the volume of which was reduced to 250 μL with apiece of Teflon. The coated electrode was inserted into the solution andincubated for 1 h. After washing (6 times) the labelled antibody in thestandard buffer (200 μL, 500 ng) was added into the cuvette andincubated again for 1 h. After the incubation the electrode was washedand the EL was measured in the same way as in Example 1, except that theEL mu g itself that was done using a side-on type photomultiplier tubeand a disposable spectophotometer cuvette made from polystyrene. Thecuvette had Teflon holder for the plate working electrode and forPt-wire counter electrode. The standard curve obtained is shown in FIG.9.

EXAMPLE 7

Immunochemical determination of β₂-microglobutin on the surface ofpulse-anodised silicon electrodes.

Silicon electrodes were made from n-Si discs doped with antimony, Theorientation of Si discs was (III) and resistivity 0.008-0.015 Ωcm(Okmetic Ltd., Finland). The electrodes were cut into the size of 8.0×55mm before surface treatments.

Anodisation of silicon electrode. Si electrode was anodised in a similarelectrolyte solution as aluminium in Example 1, but using pulseanodising with a pulse train instead of DC anodising. The pulse trainconsisted anodic and cathodic pulses (200-μs each, +5 V or −5 V,frequency 100 Hz) with 10-ms intermittent zero level between the pulses,After anodisng, the electrode was rinsed with quartz distilled water.

Coating electrode with antibody. Typically oxide-covered siliconelectrode (0.80×0.005×5.0 cm) was coated with mouseanti-β₂-microglobulin antibody (clone 6G12, Labmaster Ltd., Turku) byincubating overnight in 0.2 M NaH₂PO₄ solution, which contained 10 μg/mLof antibody. The next day the electrode %,as washed six ties withwashing solution (0.01 M TRIS-HCl buffer, pH 7.75, 0.9% NaCl and 0.02%Tween 20) and saturated overnight in a saturation solution, which was0.05 M TRIS-HCl buffer, pH 7.75, containing 1 g of bovine serum albumin,60 g of sorbitol and 1 mmol CaCl₂ per liter. The saturated electrode canbe stored dry at least for 3 months after washing (6 times).

Preparation of labelled antibody. Second monoclonalanti-β₂-microgiobulin antibody (clone 1F10, Labmaster Ltd., Turku) waslabelled with isothiosyanato derivative of theTb(II)-4[Tb³⁺-N-(4-isothiocyanatobenzoyl)-diethylentriamine-N¹, N¹, N³,N³-tetra-acetate](Wallac Ltd., Turku) by allowing the antibody to reactwith the chelate in molar ratio of 1:60 at pH 9.5. pH was adjusted with1 M Na₂CO₃ solution Labelled antibody was purified from unreactedchelate by gelfiltration (Sepharose 6B 1×50 cm, Sephadex G-50 1×5 cm)using TSA buffer (0.05 mol/L TRIS-HCl, pH 7.75, 0.9% NaCl, 0.05% NaN₃)as mobile phase. Typically in this way, 5-10 chelate molecule can bebound to one antibody molecule. To improve the stability, 0.1% of bovineserum albumin was added into the solution of labelled antibody.

β₂-Microglobulin standards. Standards (0.4, 1.6, 4.0, 8.0 and 16 mg/L)were prepared from β₂-microglobulin purified from human ascites fluid(75.5 mg/mL, Labmaster Ltd., Turku, Finland) into the TSA buffer. TSAbuffer contained 7.5% bovine serum albumin.

Immunochemical determination. Immunochemical reaction was performed indisposable polystyrene cuvettes (1 mL, Brand, Cat. Number 7590 15,Wertheim Germany), the volume of which were reduced to 250 μl by aTeflon filler. Standards were diluted 1:50 with the assay buffer (0.05mol/L TRIS-HCl, pH 7.75 containing 0.9% of NaCl, 0.05% of NaN3, 0.5% ofbovine serum albumin and 0.01% Tween 20) and were added to the bottom ofcuvette (40 μL). Then 160 μL of labelled antibody (500 ng containing 100ng of labelled and 400 ng of unlabelled antibody, clone 1F10) in theassay buffer was added. Finally, the electrode coated with antibody wasplaced into the cuvette. Immunochemical reaction was allowed to takeplace for 1 h and the electrode was washed six times with washingsolution.

Measuring of EL. EL was measured using electroluminometer and a cuvetteespecially prepared for this purpose. A Pt-wire served as counterelectrode in the cuvette. Borate buffer, 0.2 mol/L, pH 7.75, containing2 mmol/L K₂S₂O₈ was used as a measuring buffer.

The standard curve obtained is shown in FIG. 10.

EXAMPLE 8

Immunometric detection of TSH using anodised silicon electrodes andanodised silicon electrodes which were additionally coated withpolystyrene.

Coating electrodes with polystyrene. Silicon electrodes were preparedand anodised as in Example 7. The anodised electrodes were coated withpolystyrene by sonicating the electrode for 30 seconds in a solutioncontaining 1.5 mg/mL of polystyrene in benzene. After this, theelectrode was lifted up slowly from the solution and allowed to dry atroom temperature.

Electrodes were coated with antibody as in Examples 2 and 7 (clone 8661Phamacia, Uppsala, Sweden). The immunoassay was done in similar cuvettesas in Example 7, but using the labelled antibody prepared in Example 2(80 ng of Tb(III)-2 labelled antibody/electrode) and the standardsolution from Example 2. FIG. 11 shows the standard curves obtained withpotentiostatic excitation (−10 V, 200 μs excitation pulses, 500excitation cycles). Electrodes that were not anodised (a), anodisedelectrodes (by and the electrodes that were both anodised and coatedwith polystyrene (c).

EXAMPLE 9

DNA hybridisation method exploiting Si-electrodes modified byanodisation and coating with polystyrene.

Silicon electrodes were prepared from n-Si discs doped with antimony.The orientation of Si discs was (111) and resistivity 0.008-0.015 Ωcm(Okmetic Ltd., Finland). The electrodes were cut into the size of 8.0×55mm before surface treatments. Anodisation of electrodes. Electrodes wereanodised in a neutral 0.5 M ammoniumborate buffer firstgalvanostatically (1 mA/cm²) up to 5.2 V and then potentiostatically for10 minutes.

Coating with polystyrene. The anodised parts of the electrode werecoated with polystyrene at first by sonicating the electrode for 10seconds in a solution containing 10 mg/mL of polystyrene in benzene.After is the electrode was lifted up slowly from the solution andallowed to dry at room temperature.

Hybridisation and the EL measuring were carried out as in Example 4except that the electroluminometer and the cuvettes were as in Example7. The results are shown in FIG. 12.

EXAMPLE 10

Immunochemical detection of T4 with anodised Si-electrodes.

The Si-electrodes were prepared and anodised as in Example 9 and coatedwith antibody as in Example 3.

Immunochemical detection. Twenty μL of standard, 200 μL of monoclonalmouse anti-T4 antibody (Medix Biotech, Inc, USA) and 200 mLgelatin-T4-Tb-conjugate (50 ng/mL) were pipetted to a disposablecuvette. The electrode was placed into the cuvette and incubated for 1.5h with shaking and then washed 6 times. The measuring was done in as inExample 7. The standard curve is shown in FIG. 13.

EXAMPLE 11

The immunochemical determination of TSH exploiting Zn-electrodes coatedwith alternating layers of polystyrene and paraffin.

Cup electrodes (volume 450 μl) made of Zn (Johnson Matthey AlfaProducts) were first washed in an ultrasonic bath with hexane. Next,they were either treated cathodically in a peroxodisulfate solution inorder to produce a thin oxide layer, or were coated directly withorganic layers.

Cathodic oxidation of Zn-electrodes. The zinc cups were filled with0.450 ml of 0.01 M K₂S₂O_(g) solution in a 0.2 M borate buffer, pH 9.2,and catbodised with 10000 pulses (200-μs/pulse, amplitude of −10 V, andfrequency of 100 Hz). After this, the electrodes were washed with quartzdistilled water and allowed to dry.

Coating electrodes with organic layers. Oxidised or unoxidisedelectrodes were dipped slowly into a benzene solution of polystyrene(0.5 mg/mL). The electrodes were allowed to dry at room temperature for24 h. Next, the electrodes coated with polystyrene were slowly dippedinto a pentane solution of paraffin (1.0 mg/mL) and allowed to dry for 8h. Then a new polystyrene layer was added as described in Example 8.

Immunoassay was done in the same way as in Example 1 but with thereagents from Example 2. Standard curves are shown in FIG. 14.

EXAMPLE 12

Immunometric determination of β₂-microglobulin exploiting insulatorelectrodes composed from glass plates covered with indium tin oxidesurface film and coated with alternating layers of polystyrene andparaffin.

The electrodes (7.0×55 mm) were cut out from indium tin oxide-coatedglass plates (Lohja Oy, Lohja, Finland) and then coated withpolystyrene-paraffin-polystyrene layers as in Example 11.

Preparation of labelled antibody, Second monoclonalanti-β₂-microglobulin antibody (clone 1F10, Labmaster Ltd., Turku,Finland) was labelled with the isothiocyanato derivative of Tb(III)-2chelate. The labelling was done as in Example 7 with the isothiocyanatoderivative of Tb(III)-4 chelate. The label was purified by gelfiltrationas in Example 7.

Immunochemical determination and EL measurements were carried out as inExample 7. Standard curve is shown in FIG. 15.

EXAMPLE 13

Immunometric determination of β₂-microglobulin exploiting polyethyleneterephtalate sheets covered with transparent gold film (Au-PET foils)and coated with alternating layers of polystyrene and paraffin.

Au-PET foils (Intrex film, type 28FX43, Sierracin, Sylmar, Calif., USA)were glued on glass plates (8.0×55 mm) and the plate electrodes obtainedwere coated with polystyrene-paraffin-polystyrene layers as in Example7. The immunochemical detection of β₂-microglobulin was done as inExample 7, but using the labelled antibody prepared in Example 12.Standard curve is shown in FIG. 16.

EXAMPLE 14

Immunometric determination of CRP using insulator electrodes based on aconducting polymer film coated with alternating layers of paraffin andpolystyrene.

Preparation of conducting polymer layer and coating it with insulatinglayers. Steel electrodes were coated with polyaniline according to thepaper: J. Michaelson, A. McEvoy and N. Kuramoto, React. Polym. 17 (1992)197. Electrodes coated with conducting polymer were coated further withparaffin layer by dipping then into a hexane solution containing 1.0mg/mL of paraffin. The electrodes were allowed to dry for 12 hours.After this the electrodes were coated with polystyrene layer by dippingthem into a benzene solution containing 1.0 mg/mL of polystyrene.

The coating of the electrodes with antibody and immunoassays of CRP werecarried out as in Example 6. The resulting standard curve is shown inFIG. 17.

EXAMPLE 15

Immunometric determination of CRP exploiting insulator steel electrodescoated with aluminium oxide and polystyrene.

Coating steel with Aluminium oxide. Aluminium oxide layer was grown fromAl(CH₃)₃ and water on the steel plate (50×50 mm) at 200° C. by usingALE-technique. Ninety cycles that produce about 5 nm thick oxide layer,were used in growing. Before the steel plate was taken out of thereactor, it was allowed to cool down to 60° C. in 10 mbar nitrogenatmosphere.

Aluminium oxide-coated steel was cut to 7.0 mm wide strips and they werecovered with a thin polystyrene layer by dipping them twice into abenzene solution containing 0.7 mg/mL of polystyrene. The strips wereallowed to dry between and after dipping for 8 h at room temperature.

Immunoassay was done in the same way as in Example, except that TSAbuffer (pH 7.8) were the incubation buffer. Standard curve is shown inFIG. 18.

EXAMPLE 16

Detection of mouse IgG by labels emitting short- and long-lived emissionexploiting oxide-covered magnesium coated with polystyrene.

Magnesium electrodes were coated with polystyrene as described inExample 6 and coated with mouse IgG as in Example 1.

Preparation of labelled antibodies. Rabbit antibody to mouse IgG (Dako,Denmark) was labelled with fluorescein-5-isothiocyanate (Sigma, F-7250)as the antibody was labelled with Tb(III)-1-isotiocyanate in Example 1.The molar ratio of antibody to label was 1:200. Accordingly, the sameantibody was labelled with Eu(III)-3-isothiocyanate. The molar ratio ofantibody to label was 1:100.

Detection of mouse IgG. The immunreaction was made as in Example 6.Labelled antibodies were added in molar ratio of 1:01 (Eu(III)-3-labelas an excess, total amount 200 ng/mL). After 30-min incubation,electrode was washed with distilled water and transferred to a cuvetteof time-resolved spectrometer and the EL-spectra were recorded with timeslices of 500 μs (FIG. 19). nm spectrum shows that simultaneousshort-lived (fluorescein label) and long lived (Eu(III)-3-label)emission can be separated by time-resolved detection. When electrode wascoated with rabbit IgG, spectra of neither one of the labels wereobtained. The spectra were recorded as in Example 1, with a spectrometerdescribed elsewhere (S. Pihlajamäki and J. Kankare, J. Anal. Instrum. 18(1986)171.).

EXAMPLE 17

Simultaneous measurement of TSH and PLA₂ using anodisedpolystyrene-coated aluminium electrodes.

Labelling of antibodies. Monoclonal antibody specific to human TSH(clone 5404, Medix Oy, Helsinki) was labelled withaminohexylethylisoluminol (AHEI) by the method of Schroeder etal.(Methods in Enzymology, Vol 57, M. DeLuca (Ed,), Academic Press,N.Y., 1978).

Immunoassay. PLA₂ and TSH standards (25 μL of each) and 175 μL ofmixture of antibodies in TSA-buffer (500 ng anti-PLA₂-antibody and 300ng and-TSH antibody) were added into the cuvettes. Electrodes (7.0mm×0.3 mm×50 mm) were inserted into cuvettes and incubated for 1 h withshaking. Electrodes were washed with a wash solution and EL was measuredwith an electroluminometer as in Example 16. Standard curves art shownin FIG. 20.

EXAMPLE 18

Immunoassay of PLA₂ using latex particles as the solid phase carriersand polystyrene-coated anodised aluminium cups.

Aluminium electrodes were anodised and coated with polystyrene as inExample 1.

Coating of latex particles with antibodies. Stock suspension of latexparticles (Sigma, LB-8) was diluted to 1:100 with TSA buffer. Into thisdilution (100 μL), 100 μL of solution containing 5.7 mg/ml ant-PLA₂antibody (clone 2E1, Labmaster Oy, Turku, Finland) in TSA buffer and themixture was incubated overnight. Particles were separated bycentrifugation and washed with the wash solution. After this the latexparticles wet saturated with 0.1% bovine serum albumin solution as inExample 1. Tb(III)-1 chelate—antibody preparate was the same as inExample 1.

Immunoassay. About 180 million coated latex particles (20 μl of dilutionof 1:20) were applied to 1.5-mL polypropylene centrifugal tubessaturated with bovine serum albumin. To this suspension, 20 μl ofstandard and 20 μl of Tb(III)-1-labelled antibody (500 ng) were addedand incubated together for 20 min at room temperature. Then theparticles were recovered by centrifugation and washed 2 times with thewash solution and once with EL-assay solution, to which 0.02% TWEEN 40(w/v) were added. In the particles were suspended into EL measurementsolution and EL was measured by using the cup electrodes. FIG. 21 showsthe standard curve obtained.

EXAMPLE 19

Enzymatic amplification in an immunoassay of PLA₂ with latex particlesas the solid carriers exploiting polystyrene-coated magnesiumelectrodes.

Magnesium electrodes were coated with polystyrene film as in Example 6.Latex particles were coated with antibodies as in Example 18.

Labelling of antibodies with alkaline phosphatase. Polyclonal sheepanti-PLA₂ was labelled with alkaline phosphatase (ALF) with maleimide(E. Ishikawa, M. Imagawa, S. Hashida, S. Yoshitake, Y. Hamuguchi and T.Ueno, J. Immunoassay, 4 (1983) 209.).

Immunoasay. About 180 million coated latex particles (20 μl of dilutionof 1:20) were applied to 1.5-mL polypropylene centrifugal tubessaturated with bovine serum albumin. To this suspension, 20 μL ofstandard and 20 μl of ALF-labelled antibody (600 ng) were added and themixture was incubated for 15 min at room temperature. Then the particleswere separated by centrifugation and washed 2 times with the washsolution. Particles were suspended into 200 μl of a solution containing1 mmol/L substrate (phosphoric acid ester of 5-fluorosalisylic acid,FSAF, Kronern Systems Inc. Mississauga, Ontario Canada). Thesupernantant was separated after a 15-min incubation and 100 μL of itwas transferred into EL-cuvette. It contained a disposable coatedMg-working electrode and 2 non-disposable platinum wires as the counterelectrode. Then 150 μl of 0.1 M NaOH containing 1.0 mmol/L potassiumperoxodisulfate were added. EL was measured though a 420-nm interferencefilter. The standard curve (a) is shown in FIG. 22. An alternativedetection method was the following: 150 μL of supernatant was added to100 μL of 0.5 mM Tb(III)-EDTA solution in 0.2 M NaOH. The solution wasmixed with a pipette and allowed to react for 15 min. The solution wastransferred to EL cuvette and 25 μL of 0.01 M potassium peroxodisulfatewere added. After mixing with a pipette, EL-signal was measured.Standard curves are shown in FIG. 22. Both total (b) and time-resolved(c) signals (8 ms window and 50 μs delay) were measured through 545 nminterference filter by integrating over 10000 excitation pulses.

EXAMPLE 20

Immunoassay for β₂-microglobulin on microtiter strips by detaching Tbions from antibodies after the immunoreaction and measuring free Tb ionswith a new complex using anodised silcon electrodes.

The wells of microtiter strips were coated as described in Example 7 forthe electrodes. The antibody was labelled with T(III)-1 chelate as inExample 7 and the immunoassay was performed also as in Example 7. After1-h incubation, the wells were washed with the wash solution and 200 μLof 0.1 M glycine-H₂SO₄-buffer, pH 2.5, were added followed by a 15-minincubation. Then 150 μL of the incubated solution was taken into thecuvette and 45 μL of 0.5 mol/L Na₂CO₃ containing 5×10⁻⁴ mol/L of ligand5 was added The solution was mixed well and 205 μL of measuring bufferwas adder The Tb(III)-5 chelate thus produced was quantified withoxide-coated silicon electrodes as in Example 7. The standard curve isshown in FIG. 23.

EXAMPLE 21

Immunoassy for β₂-microglobulin with liposomes as carriers of the labelexploiting anodised silicon electrodes.

Liposomes containing Tb-5-complex were prepared. They were bound toantibodies (anti-β₂-microglobulin, clone 6G12, Labmaster Oy, Turku,Finland) according to OP. Vonk, B. Wagner, Clin. Chem., 37 (1991) 1519.The immunoreaction was made as in Example 20, except that the buffer didnot contain detergents. After washing, 230 μL of 0.1% Triton X-100, pH3.2, in 0.1 M glycin buffer were added and incubated for 10 min. To thissolution (200 μL), 60 μL of 0.5 mol/L Na₂CO₃ containing 5×10⁻⁴ mol/L ofligand 5, were added Measuring buffer (240 μL) was added to the cuvetteand EL was measured using 5 V (DC) anodised silicon electrodes as inExample 20. Standard curves are shown in FIG. 24.

EXAMPLE 22

Immunoassay for β₂-microglobulin employing UV-photochemical detaching ofthe label followed by measurement using anodised silicon electrodes.

Microtiter strips were coated as in Example 20.

Labelling of antibodies. The antibody (clone 1F10, Labmaster Oy, Turku,Finland) was labelled with a photodetachable label (Rhodamine Givensulfosuccinimidyl ester, Molecular Probes, R-7091, Eugene, USA)according to the manufacturer's instructions.

The immunoassay was carried out in the wells of microtiter plates as inExample 20, except that after the washing, 200 μL of 0.05 M Na₂B₄O₇ werepipetted into the wells.

Detaching the label with UV-radiation and EL-measurement. Microtiterstrips were exposed to UV-light (Philips HPLR) for 4.0 min from the top.From each well, 180 μL of solution was taken and transferred to acuvette and 320 μL of measuring solution were added. Total emission wasmeasured as in Example 20 but with using a band-pass filter of 520 nm.Standard curve is shown in FIG. 25.

EXAMPLE 23

Homogeneous immunoassay for β₂-microglobulin exploiting energy transferfrom donor to acceptor, enabling to observe delayed light emission fromacceptor with an life-time related to the emission life-time of thedonor.

Labelling of antibodies. Coating antibody (clone 6G12, Labmaster Oy,Turku, Finland) was labelled with Tb(III)-2-isothiocyanate as theLabelling of anti-TSH antibody in Example 2. Second monoclonal antibody(clone 1F10, Labmaster Oy, Turku, Finland) was labelled with Rhodamine Bisotiocyanate (Sigma, R 1755) using label/antibody ratio of 90:1.Rhodamine B-labelled antibody was diluted after purification into an 0.2M borate buffer, pH 7.8, including 4% bovine serum albumin.

Coating of electrodes. Silicon electrodes were coated withTb(III)-2-labelled antibodies as were done in Example 7 with thenon-labelled antibody.

Immunoassay. Standard solution (40 μL; the same standards as in Example7 in dilution of 1:50 made into 0.2 M borate buffer, pH 7.8, containing4% of bovine serum albumin and 0.005% Tween 20) and 160 μL of RhodamineB-labelled antibody (800 ng) were applied into the cuvette. Electrodewas inserted into the cuvette containing disposable steel counterelectrodes. After a 20-min incubation wit shaking, the cuvette wastightly closed and placed into the electroluminometer. The ratio of ELsignal at 490 nm and 670 nm (delay time 500 μs; measuring window 3.0 ms)was measured. Results are in FIG. 26.

EXAMPLE 24

Homogeneous immunoassay for β₂-microglobulin usingterbium(III)-chelate—containing latex particles and anodised siliconelectrodes.

Preparation of latex particles and coating with antibodies. Into asaturated solution of Tb(III)-5 chelate in benzene, polystyrene wasdissolved to obtain concentration of 20 mg/mL. This mixture (400 μL) waspipetted into 15 mL of pentane in an ultrasonic bath and sonicated for 5min. Water (15 mL) was added and the mixture was shaken for 2 min. Thelatex parties were separated by centifugation and suspended in 0.5 mL ofTSA buffer.

Labelling of antibodies. The coating antibody (clone 6G12, Labmaster Oy,Turku, Finland) was labelled with 9-fuorenylmethylclhoroformate (FMOC,Aldrich, No. 16,051-2) according the manufacturer's instructions withusing label/antibody ratio of 80:1.

Coating of electrodes. Silicon electrodes we prepared and anodised as inExample 9. They were coated with FMOC-labelled antibody as was done withunlabelled antibody in Example 7.

Coating of latex particles. Latex particles were coated with antibodies(Clone 1F10, Labmaster Oy, Turku, Finland) as in Example 10.

Immunoassy. Standard solution (40 μL; the same standard solutions as inExample 7 as a dilution of 1:50 made up by 0.2 M borate buffer, pH 7.8,including 4% bovine serum albumin and 0.005% Tween 20), 160 μL of theformer buffer containing 1 mmol/L K₂S₂O₈ and 20 μL of the particlesuspension were mixed in the cuvette. The reaction was allowed toproceed with shaking for 20 min and then 10 min without shaking.Eventually, EL was measured as in Example 7. Standard curve is shown inFIG. 27.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 2 <210> SEQ ID NO 1 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:      oligonucleotide containing amino groups <400> SEQUENCE: 1agatgctact ggccgctgaa gggctt           #                  #              26 <210> SEQ ID NO 2 <211> LENGTH: 28 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial  #Sequence:      oligonucleotide containing an amino  #group <400> SEQUENCE: 2ttccttattg atggtcagcg gaatgctg          #                  #             28

What is claimed is:
 1. A method for electrical excitation of a labelmolecule, comprising at least partially immersing an electrode in anelectrolyte solution containing at least one label molecule; excitingsaid label molecule by an electrical pulse from said electrode, therebyproducing an excited label; and detecting luminescence emitted by saidexcited label; wherein said electrode comprises an electricallyconductive material and at least the portion of said electrode which isimmersed in said solution is substantially covered with an electricallyinsulating film comprising aluminum oxide having a band gap equal to orgreater than 5 eV, wherein said electrically insulating film is coveredby a shielding layer comprising an organic polymeric material.
 2. Themethod of claim 1, wherein the electrically conductive material of saidelectrode has a resistivity below 10 Ωcm.
 3. The method of claim 2,wherein said electrically conductive material is a member selected fromthe group consisting of metal, a conductive polymer, and an electricallyconductive doped semiconductive material.
 4. The method of claim 3,wherein said electrically conductive material is silicon or compositesilicon made to be conductive by doping with other materials.
 5. Themethod of claim 1, wherein said electrode is sufficiently transparent ata wavelength range of emission of said label that luminescence can bemeasured through said electrode.
 6. The method of claim 1, wherein saidlabel is a part of a detectable molecule which conforms to the formulaL_(n)—X_(x)—Y_(y), wherein L is said label; X is a linking compound; Yis a compound having affinity against the analyte to be quantified, andis a member selected from the group consisting of proteins, antibodies,enzymes and nucleic acids, and wherein the integral subscripts n, x, andy express the number of L, X, and Y, and are equal to or larger than 1.7. The method of claim 6, wherein said compound Y is capable of bindingto at least one member of the group consisting of a cell, a cellcomponent, a virus, a bacterium, a nucleic acid, a DNA, a RNA, aDNA-fragment, a RNA-fragment, a polysaccharide, a protein, apolypeptide, an enzyme, a metabolite, a hormone, an alkaloid, a steroid,a vitamin, an amino acid, a carbohydrate and an antibody.
 8. The methodof claim 6, wherein X comprises a chemical linking functional groupselected from the group consisting of ureido, thioureido, amide,substituted imide, thioether, —S—S—, sulfonamide and N-substitutedsulfonamide, and wherein said chemical functional linking group is apart of a larger molecule or polymer attached to Y.
 9. The method ofclaim 6, wherein said label is a derivative of a compound selected fromthe group consisting of an organic luminophore derivative, a derivativeof a lanthanide chelate and a derivative of a transition metal chelate.10. The method of claim 9, wherein said organic luminophore is selectedfrom the group consisting of fluorescein, aminonaphthalenesulphonicacid, salicylates, rhodamines and coumarines.
 11. The method of claim 9,wherein said lanthanide chelate is a member of the group consisting ofTb(III), Eu(III), Y(III), Sm(III), Dy(III) and Gd(III) chelates.
 12. Themethod of claim 9, wherein said transition metal chelate derivative is aderivative of ruthenium(II)-trisbipyridyl or ruthenium(II)-trispyrazyl.13. The method of claim 6, wherein said electrode further comprises acompound Y′ bound directly or indirectly to a surface of said electrode,said compound Y′ having a specific affinity to the same analyte as Y,and wherein an affinity reaction takes place on a surface of theinsulating film before said detecting step.
 14. The method of claim 6,wherein a competition of binding to Y on a surface of said electricallyinsulating film is created between a labeled analyte which conforms tothe formula L_(n)—X_(x)—A_(a) wherein A is an analyte of interest, andthe integral subscripts n, x and a express the number of L, X and A. 15.The method of claim 14, wherein binding of L_(n)—X_(x)—Y_(y) to A on asurface of said electrically insulating film is inhibited by Aoriginating from a sample.
 16. The method of claim 6, further comprisinga washing step which removes free label L_(n)—X_(x)—Y_(y) from theproximity of said electrode before said detecting step.
 17. The methodof claim 6, further comprising an incubation step in which saiddetectable molecule is bound to a solid support in the form of particlesprior to being excited.
 18. The method of claim 17, wherein aluminophore is detached from the detectable molecule bound to a solidsupport before quantification of the luminescence signal.
 19. The methodof claim 6, wherein said label L is an enzyme capable of amplificationof luminescent luminophores.
 20. The method of claim 19, wherein saidenzyme is alkaline phosphatase, and the luminescent molecule is alanthanide chelate generated by said enzyme.
 21. The method of claim 1,wherein more than one kind of label is used simultaneously.
 22. A methodaccording to claim 1, further comprising adding at least one coreactantcapable of transforming at least one primary reducing or oxidizingcomponent generated by said electrode, such that said at least oneprimary or oxidizing component is transformed into a secondary oxidizingor reducing component more suitable for exciting said label.
 23. Themethod of claim 22, wherein said coreactant is selected from the groupconsisting of peroxodisulfate, peroxodiphoshate and hydrogen peroxide.24. An insulating film-coated electrode, comprising an electricallyconductive material, an electrically insulating film comprising aluminumoxide covering substantially all of said electrically conductivematerial, said film having a band gap equal to or greater than 5 eV; anda compound bound directly or indirectly to a surface of said electrode,said compound is capable of binding to a member of the group consistingof a cell, a cell component, a virus, a bacterium, a nucleic acid, aDNA, a RNA, a DNA-fragment, a RNA-fragment, a polysaccharide, a protein,a polypeptide, an enzyme, a metabolite, a hormone, an alkaloid, asteroid, a vitamin, an amino acid, a carbohydrate and an antibody;wherein said electrically insulating film is covered by a shieldinglayer comprising an organic polymeric material.
 25. The insulatingfilm-coated electrode of claim 24, wherein said electrode issufficiently transparent that luminescence can be measured through saidelectrode.
 26. The insulating film-coated electrode of claim 24, whereinsaid electrically conductive material is silicon or composite siliconmade to be conductive by doping with other materials.
 27. The insulatingfilm-coated electrode of claim 24, wherein said electrically insulatingfilm covering said electrically conductive material has a band gapgreater than 5 eV.
 28. The insulating film-coated electrode of claim 24,wherein said electrically insulating film has an average thickness offrom 2 to 5 nm.